Anti-cobra toxin antibody fragments and method of producing a vhh library

ABSTRACT

A method of constructing a V H H library from an immunized camelid, using whole venom or an extract thereof There is also provided V H H antibody fragments isolated from a library produced in hyperimmunized llama These V H H antibody fragments were sequenced, and specifically bind α-cobratoxin.

This disclosure claims the benefit of U.S. provisional application Ser.No. 61/120,208, filed Dec. 5, 2008 and U.S. provisional application Ser.No. 61/145,667, filed Jan. 19, 2009, both of which are incorporatedherein by reference in their entirety.

FIELD

The disclosure relates to V_(H)H antibody fragments that bind to a toxinin venom and a recombinant V_(H)H library against venom.

BACKGROUND

Snake bite is a serious global public health problem, especially intropical and subtropical countries (Chippaux 1998). It is estimated thatover 5 million snake bite cases occur worldwide each year, of which 2.6million cause envenomation, and about 125,000 of these result in death(Chippaux 2006). Naja kaouthia (That cobra), a member of the Elapidaefamily, is one of the most venomous and dangerous snakes of Thailand,causing the highest mortality and morbidity due to snake envenomation(Viravan et al. 1986).

Envenomation by N. kaouthia is usually manifested by neurotoxicity andextensive local tissue necrosis (Viravan et al. 1986). The most toxiccomponent of N. kaouthia venom is α-cobratoxin, a low molecular weight(7.8 kDa) post-synaptic α-neurotoxin (Karlsson 1979 cited in Pratanaphonet al. 1997). α-Cobratoxin binds with high affinity and specificity tothe nicotinic acetylcholine receptors (nAChRs) on post-synapticmembranes of skeletal muscles, thereby preventing the access of ACh tothe receptor's binding pocket (Bourne et al. 2005). Consequently,neuromuscular transmission is blocked and symptoms of muscle flaccidparalysis result. Lethality is generally a result of respiratory failure(Minton 1990). The venom of N. kaouthia also contains several cytotoxinsthat exhibit cytotoxic activities on many cell types (Inoue et al.1987). Phospholipases A2 and metalloproteinases are the main componentsof the venom responsible for local tissue necrosis (Gutierrez et al.2000).

Antivenoms are currently the only specific treatment for snake bites.Conventional antivenoms are prepared by hyper-immunizing an animal,generally a horse, with snake venom to generate high affinity antibodiesagainst the foreign snake toxins. Horse serum is collected, and wholeIgG molecules (150 kDa) are purified and produced into F(ab′)₂ antibodyfragments (100 kDa) by pepsin digestion and/or Fab antibody fragments(50 kDa) by papain digestion (Lalloo and Theakston 2003). These antibodyfragments can then be administered intravenously to an envenomed patientto neutralize the activity of the snake venom toxins. Systemicenvenomation is generally treated efficaciously with antivenom;administration of antivenom rapidly neutralizes neurotoxicity caused bythe action of post-synaptic neurotoxins (Warrell 1992 cited in Gutierrezet al. 2006). However, antivenoms are ineffective in treating localeffects on tissues near the snake bite because of the rapid activity ofthe toxins at the local tissue, and the inability of antivenomimmunoglobulin fragments to reach and penetrate deep tissues (Gutierrezet al. 1998). Although many survive envenomation, a large number ofvictims are left with chronic physical disability and psychologicalsequelae as a result of the cytotoxic components of the snake venom(Viravan et al. 1992).

Aside from conventional IgGs, camels and llamas have evolved uniqueheavy chain IgG immunoglobulins naturally devoid of light chains and theCH1 domains (Hamers-Casterman et al. 1993). The antigen binding sites ofthese heavy chain IgGs are composed of a single variable domain (calledV_(H)Hs), and are the smallest natural antigen binding domain (15 kDa).V_(H)H antibody fragments have several properties that potentially wouldmake them superior candidates for antivenom development overconventional antivenoms. They are relatively non-immunogenic, soluble,stable, and highly tissue penetrable (Arbabi Ghahroudi et al. 1997;Cortez-Retamozo et al. 2002 and 2004; Muruganandam et al., 2002). Owingto their low molecular mass, V_(H)H antibody fragments permeate tissuecompartments more readily than conventional antibody fragments(Cortez-Retamozo et al., 2002 and 2004) and, therefore, may protectvictims from the tissue-damaging effects of venom toxins. Furthermore,because of their small size and high homology to the human V_(H)3 genefamily, V_(H)Hs may produce less adverse reactions in patients thanconventional antivenoms (Vu at al., 1997). Furthermore, V_(H)H antibodyfragments can easily be expressed and purified from E. coli/yeastexpression systems, solving the current short supply and high costcrisis of antivenoms (Arbabi Ghahroudi et al. 1997; Frenken et al.2000).

Recently, three V_(H)Hs specific for α-cobratoxin were isolated from anaïve (synthetic) llama phage display library (Stewart et al. 2007).However, the affinities of these V_(H)Hs were too low (in uM range) fortherapeutic efficacy. Therefore, there is a need in the art to obtainhigher affinity V_(H)Hs against venom such as snake venom.

SUMMARY

A phage-displayed V_(H)H library was constructed from a llamahyperimmunized with crude Naja kaouthia venom to obtain higher affinitybinders to α-cobratoxin. After three rounds of panning againstalpha-cobratoxin, 26 unique clones were determined by monoclonal phageELISA and DNA sequencing. Analyses of predicted amino acid sequencessuggest two major groups of antibodies. Surface plasmon resonance (SPR)analyses showed that 4 soluble anti-alpha-cobratoxin V_(H)Hs clones haddissociation constants (K_(D)) in the low nanomolar range (0.4-25nanoM), and that these four V_(H)Hs bound to the same or overlappingepitopes on α-cobratoxin. An in vitro muscle twitch assay showed thatV_(H)H C2 (K_(D)=0.4 nM) effectively neutralized the paralytic effectsof α-cobratoxin at neuromuscular junctions. Thus the present inventorshave generated and identified novel high affinity V_(H)H antibodyfragments against snake neurotoxins useful as novel therapeutic agentsfor the treatment of snake envenomation. The inventors also identifiedthe heavy chain complementarity determining regions (CDRs) of the V_(H)Hantibody fragments disclosed herein. The inventors further compared theamino acid sequences of the V_(H)H antibody fragments disclosed hereinand determined consensus sequences showing conserved regions.

Accordingly, in one embodiment, the disclosure provides a method ofobtaining a V_(H)H library comprising:

(1) immunizing a camelid with whole venom or an extract thereof;

(2) isolating nucleic acid sequences encoding the variable heavy(V_(H)H) fragment from the immunized camelid; and

(3) transforming a suitable host with the nucleic acid sequences toprepare a recombinant V_(H)H library comprising V_(H)H antibodyfragments that can bind to one or more proteins in the venom.

In another embodiment, the disclosure provides an isolated V_(H)Hantibody fragment that can bind to one or more proteins present invenom.

The isolated V_(H)H may be prepared by panning a recombinant V_(H)Hlibrary with one or more proteins or toxins from the venom and selectingV_(H)H fragments that bind thereto.

In another embodiment, the disclosure provides an isolated heavy chaincomplementarity determining region 1 (CDR1) comprising an amino acidsequence selected from SEQ ID NOS:5-8 and 31 or a variant thereof. Inanother embodiment, the disclosure provides an isolated heavy chaincomplementarity determining region 2 (CDR2) comprising an amino acidsequence selected from SEQ ID NOS:9-10, 18, 30 and 33 or a variantthereof. In another embodiment, the disclosure provides an isolatedheavy chain complementarity determining region 3 (CDR3) comprising anamino acid sequence selected from SEQ ID NOS:11-13, 29 and 32 or avariant thereof.

Another aspect of the present disclosure provides an isolated V_(H)Hantibody fragment comprising: one or more isolated CDR1 sequencesselected from SEQ ID NOS:5-8 and 31; and/or one or more isolated CDR2sequences selected from SEQ ID NOS:9-10, 18, 30 and 33; and/or one ormore isolated CDR3 sequences selected from SEQ ID NOS:11-13, 29 and 32;or a variant thereof. In another embodiment, the disclosure provides anisolated V_(H)H antibody fragment comprising an amino acid sequenceselected from SEQ ID NOS:1-4 or a variant thereof.

Another aspect of the present disclosure is an isolated nucleic acidsequence encoding: the heavy chain complementarity determining region 1(CDR1) comprising an amino acid sequence selected from SEQ ID NOS:5-8and 31; and/or the heavy chain complementarity determining region 2(CDR2) comprising an amino acid sequence selected from SEQ ID NOS:9-10,18, 30 and 33; and/or the heavy chain complementarity determining region3 (CDR3) comprising an amino acid sequence selected from SEQ IDNOS:11-13, 29 and 32; or a variant thereof.

The present disclosure also includes an isolated nucleic acid sequenceencoding the V_(H)H antibody fragment comprising an amino acid sequenceselected from SEQ ID NOS:1-4 or a variant thereof. Another embodiment ofthe present disclosure is an isolated nucleic acid sequence encoding theV_(H)H antibody fragment comprising a nucleic acid sequence selectedfrom SEQ ID NOS:14-17 or a variant thereof.

In one embodiment, the CDRs and/or V_(H)H antibody fragments disclosedherein bind to one or more proteins in a venom.

The disclosure includes all uses of the isolated CDRs and/or V_(H)Hfragments disclosed herein including their use in therapy to treat asubject exposed to a venom and/or to treat envenomation in a subjectand/or to neutralize a venom in a subject exposed to the venom.

Another aspect of the present disclosure is a pharmaceutical compositioncomprising an effective amount of the CDRs and/or V_(H)H fragmentsdisclosed herein with a diluent or carrier and uses of thepharmaceutical composition thereof.

The present disclosure also includes a kit comprising an effectiveamount of the V_(H)H fragments disclosed herein together with ancillaryagents and instructions for the use thereof.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the disclosure are given by wayof illustration only, since various changes and modifications within thespirit and scope of the disclosure will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1; (a) Conventional IgG (cIgG), (b) Heavy-chain antibody (HCAb) ofcamelids, and (c) V_(H)H antibody fragment.

FIG. 2: Western blot analysis of soluble α-Cbtx V_(H)H clones.

FIG. 3: Surface plasmon resonance analysis of immobilized α-Cbtx v.santi-α-Cbtx V_(H)H C2, C19, C20 and C43.

FIG. 4: Epitope competition using surface plasmon resonance. Aftersaturation of α-Cbtx with C19 (a) or C43 (b), the other three V_(H)Hswere injected (arrows). An increase in RU indicates further binding toα-Cbtx.

FIG. 5: Attenuation of α-Cbtx inhibition of the tetanic response tophrenic nerve stimulation by V_(H)H. Tetanic response in an untreatedpreparation (top line), following α-Cbtx 50 nM plus V_(H)H 100 nM(middle line), and following 120 min later α-Cbtx 50 nM without V_(H)H(bottom line). Pretreatments were for 30 min. α-Cbtx was co-incubatedwith V_(H)H for 60 min at room temperature prior to adding to the tissuebath.

FIG. 6: Predicted amino acid sequence alignment of anti-α-Cbtx V_(H)Hbinders isolated from the 3rd round of panning. The clones werecategorized with either Cluster I or Cluster II based on their sequencehomology. Residues are numbered according to the Kabat numbering system(Kabat and Wu, 1991). The dots in the sequences indicate amino acididentity that is the same as in C33 (Cluster I) or C2 (Cluster II). Allclones belong to V_(H)H Subfamily 2 (Harmsen et al., 2000). An asterisk(*) represents an amber stop codon (TAG) mutation.

DETAILED DESCRIPTION

As previously mentioned, the present inventors have constructed a V_(H)Hlibrary from a llama hyperimmunized with N. kaouthia venom and isolatedV_(H)H clones with high affinity and specificity to α-cobratoxin byphage display technology. The isolated V_(H)H clones were shown toneutralize the toxin in an in vitro mouse muscle twitch assay.

I. V_(H)H Library

In one embodiment, the disclosure provides a method of obtaining aV_(H)H library comprising:

(1) immunizing a camelid with whole venom or an extract thereof;

(2) isolating nucleic acid sequences encoding the variable heavyfragment (V_(H)H) from the immunized camelid; and

(3) transforming a suitable host with the nucleic acid sequences toprepare a recombinant V_(H)H library comprising V_(H)H antibodyfragments that can bind to one or more proteins in the venom.

The term “V_(H)H” as used herein means the variable domain of a heavychain antibody isolated from a camelid. A V_(H)H is shown schematicallyin FIGS. 1 b) and c).

The term “camelid” as used herein means a member of the family Camelidaeincluding, without limitation, llamas, camels, dromedaries, alpacas,vicunas and guanacos. In one embodiment, the camelid is a llama or acamel.

The term “venom” as used herein means a substance that is released froman animal in order to immobilize, kill or facilitate digestion of itsprey. Venoms are comprised of a mixture of enzymes, toxins, proteins andother small molecules. Animals that release a venom include, withoutlimitation, snakes, spiders, scorpions, centipedes, stinging insects(such as bees or wasps), fish (such as stingrays and sharks), jellyfish, gila monster and Mexican lizards. In a specific embodiment, thevenom is a snake venom.

The term “extract” of venom includes one or more of the enzymes, toxins,proteins or small molecules present in the complete venom. Examples ofcomponents of snake venom can be found in Table 4.

The term “nucleic acid sequences” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentdisclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine.

The camelid can be immunized with the venom or extract thereof at leastonce, although in one embodiment at least one booster immunization isgiven. For the initial immunization, the camelid may be injected at morethan one site, or may be injected at two sites, or alternatively, may beinjected at three sites. After each immunization regime, the antibodytiter will be checked. Immunization will continue until antibody titerlevels reach a plateau indicating the animal is hyperimmunized.

Once the desired antibody titer is reached, nucleic acid molecules, forexample mRNA can be purified from lymphocytes of the hyperimmunizedcamelid. The mRNA transcripts can be reverse transcribed into a libraryof cDNA. From this library, V_(H)H DNA fragments, encoding the variableheavy fragment of the heavy-chain antibody, can be PCR amplified using5′-forward and 3′-reverse primers complementary to ends of the V_(H)Hdomain, making a repertoire of V_(H)H genes. In order to clone theseV_(H)H fragments into a phage vector, restriction sites can beintroduced by PCR, for example using primers with Sfi I restrictionsites flanking the V_(H)H sequence. After digestion with appropriatenuclease, the repertoire of V_(H)H fragments will be ligated with phagevector and transformed into competent host strain by electroporation.The host can be any suitable host including, without limitation,bacteria, yeast, plant cells and animal cells. In one embodiment, thehost is a bacteria such as E. coli.

The term “primer” as used herein refers to a nucleic acid sequence,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofsynthesis of when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand isinduced (e.g. in the presence of nucleotides and an inducing agent suchas DNA polymerase and at a suitable temperature and pH). The primer mustbe sufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon factors, including temperature, sequences of theprimer and the methods used. A primer typically contains 15-25 or morenucleotides, although it can contain less. The factors involved indetermining the appropriate length of primer are readily known to one ofordinary skill in the art.

The term “complementary” refers to nucleic acid sequences capable ofbase-pairing according to the standard Watson-Crick complementary rules,or being capable of hybridizing to a particular nucleic acid segmentunder stringent conditions.

The phage-display library can be used to select V_(H)H antibodyfragments that bind, with high affinity and specificity, to venomtoxins. In this process, transformants harboring recombinant phagevector can be infected with M13 helper phage K07 (KM13) to produce apopulation of phage particles, each displaying a unique V_(H)H antibodyfragment. V_(H)H clones specific for a particular toxin in the venom maybe selected. In one embodiment, the library may be panned againstseveral venom toxins. For example, phage particles may be panned againstboth immobilized α-cobratoxin and an immobilized mixture of crude venomtoxins, separately. Phage particles carrying V_(H)H antibody fragmentsspecific for the antigen will bind, and unbound phages will be washedaway. Bound phages will be eluted, propagated in E. coli by helper phageinfection, and used for the next round of panning. Each consecutiveround of panning should enrich the antigen-binding specificity ofclones. Generally, three to four rounds of panning are performed toselect antibody fragments with desired affinity. The disclosure includesall V_(H)H antibody fragments isolated from the library disclosedherein. In one embodiment, the V_(H)H antibody fragment isolated fromthe library can bind to one or more proteins and/or toxins present in avenom. In another embodiment, the venom is a snake venom. In yet anotherembodiment, the V_(H)H antibody fragment isolated from the library bindsto α-cobratoxin.

II. Complementarity Determining Regions and V_(H)H Antibody Fragments

As mentioned above, the present inventors have identified novel highaffinity V_(H)H antibody fragments. The nucleotide and amino acidsequences are shown in Table 5. The inventors also identified the heavychain complementarity determining regions (CDRs) of the V_(H)H antibodyfragments (See Table 2), and also determined a consensus sequenceshowing a conserved region of the V_(H)H antibody fragments disclosedherein (See Table 7). The inventors analyzed the amino acid sequencealignment of V_(H)H antibody fragment clones isolated after the 3^(rd)round of panning, which were categorized as Cluster I or Cluster IIbased on their sequence homology, and further identified consensusmotifs for CDR1, CDR2 and CDR3 in Clusters I and II (see FIG. 6).

Accordingly, in one embodiment, the disclosure provides an isolatedheavy chain complementarity determining region 1 (CDR1) comprising theamino acid sequence: GSISSIYAMG (SEQ ID NO:5); GSTFDDYAIG (SEQ ID NO:6);GDISSFNAMG (SEQ ID NO:7); or GSISSFNGMG (SEQ ID NO:8); an isolated heavychain CDR2 comprising the amino acid sequence: VITNGNSPNYADSVKGR (SEQ IDNO:9); or FISSGGRSKYTDSVKGR (SEQ ID NO:10); and an isolated heavy chainCDR3 comprising the amino acid sequence: EGVRYGDSWYDGDY (SEQ ID NO:11);GSWSYETGNYYEPSNY (SEQ ID NO:12); or GSVLSYVTGNYYEPSDY (SEQ ID NO:13). Asnoted above, the inventors have determined a consensus sequence for theV_(H)H antibody fragments disclosed herein. In particular, the consensussequence comprises the amino acid sequence: DSVKGRFTIS (SEQ ID NO:18)and occurs within CDR2 of the V_(H)H antibody fragments disclosedherein. Accordingly, in one embodiment, CDR2 comprises the amino acidsequence in SEQ ID NO:18.

As noted above, the inventors determined consensus motifs for CDR1, CDR2and CDR3 for clones categorized as Cluster I or II. In particular, theconsensus motif for CDR1 in Cluster I comprises the amino acid sequence:G(D/S)ISSFN(A/G)MG) (SEQ ID NO:31); the consensus motif for CDR2 inCluster I comprises the amino acid sequence: FISSGGRSKYTDSVK; (SEQ IDNO:30); the consensus motif for CDR3 in Cluster I comprises the aminoacid sequence: GSV(V/L/I)SY(E/V)TGNYYEPS(N/D)Y (SEQ ID NO:29); theconsensus motif for CDR1 in Cluster II comprises the amino acidsequence: GSISSIYAMG (SEQ ID NO:5); the consensus motif for CDR2 inCluster II comprises the amino acid sequence: VITNGNSPNYADSVKG (SEQ IDNO:33); and the consensus motif for CDR3 in Cluster II comprises theamino acid sequence: EGVRYGDSWYDG(D/V)Y (SEQ ID NO:32). Accordingly, inanother embodiment, CDR1 comprises the amino acid sequence in SEQ IDNO:31; CDR2 comprises the amino acid sequence in SEQ ID NOS: 30 and 33;and CDR3 comprises the amino acid sequence in SEQ ID NOS: 29 and 32. Inyet another embodiment of the present disclosure, the isolated CDR1,CDR2, and/or CDR3 comprising the amino acid sequences in SEQ ID NOS:5-8,and 31; SEQ ID NOS:9-10, 18, 30 and 33; and/or SEQ ID NOS:11-13, 29 and32; respectively, can bind to one or more proteins present in venom.

The term “heavy chain variable region” as used herein refers to thevariable domain of a heavy chain.

The term “heavy chain complementarity determining region” as used hereinrefers to regions of hypervariability within the heavy chain variableregion of an antibody molecule. The heavy chain variable region hasthree complementarity determining regions termed heavy chaincomplementarity determining region 1, heavy chain complementaritydetermining region 2 and heavy chain complementarity determining region3 from the amino terminus to carboxy terminus.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified amino acids.

Additional aspects of the present disclosure are isolated V_(H)Hantibody fragments comprising one or more of the isolated heavy chainCDR1, CDR2 and/or CDR3 of the present disclosure (SEQ ID NOS:5-13, 18,and 29-33). In one embodiment, the isolated V_(H)H antibody fragmentcomprises the isolated heavy chain CDR1, CDR2 and CDR3 selected from SEQID NOS:5, 9 and 11, respectively. In another embodiment, the isolatedV_(H)H antibody fragment comprises the isolated heavy chain CDR1, CDR2and CDR3 selected from SEQ ID NOS:6, 9 and 11, respectively. In anotherembodiment, the isolated V_(H)H antibody fragment comprises the isolatedheavy chain CDR1, CDR2 and CDR3 selected from SEQ ID NOS:7, 10 and 12,respectively. In another embodiment, the isolated V_(H)H antibodyfragment comprises the isolated heavy chain CDR1, CDR2 and CDR3 selectedfrom SEQ ID NOS:8, 10 and 13, respectively. In one embodiment, theisolated V_(H)H antibody fragments comprising the isolated heavy chainCDR1, CDR2 and/or CDR3 of the present disclosure (SEQ ID NOS:5-13, 18,and 29-33) can bind to one or more proteins present in venom.

In another embodiment of the present disclosure, the V_(H)H antibodyfragment comprises the amino acid sequence:QVKLEESGGGLVLPGGSLRLSCAASGSISSIYAMGWYRQAPGKQREVVAVITNGNSPNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVEGVRY GDSWYDGDYWGQGTQVTVSS(SEQ ID NO:1). In another embodiment, the V_(H)H antibody fragmentcomprises the amino acid sequence:QVKLEESGGGLAQAGGSLRLSCIGSGDISSFNAMGWYRQVPGKQRELVAFISSGGRSKYTDSVKGRFTISGDNAKNTVYLQMIDLKPEDTAVYYCNAGSVVSYETGNYYEPSNYWGQGTQVTVSS (SEQ ID NO:2). In another embodiment, the V_(H)Hantibody fragment comprises the amino acid sequence:QVKLEESGGGLVQPGGSLRLSCVGSGSISSFNGMGWYRQVPGKQRELVAFISSGGRSKYTDSVKGRFTISGDNAKSTVYLQMINLKPEDTAVYYCNVGSVLSYVTGNYYEPSDYWGQGTQVTVSS (SEQ ID NO:3). In a further embodiment, theV_(H)H antibody fragment comprises the amino acid sequence:RVKLEESGGGLVQAGGSLRLSCAVSGSTFDDYAIGWYRQAPGKQREVVAVITNGNSPNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVEGVRY GDSWYDGDYWGQGTQVTVSS(SEQ ID NO:4). In another embodiment, the isolated V_(H)H antibodyfragments comprising the amino acid sequences selected from SEQ IDNOS:1-4 can bind to one or more proteins present in venom.

The present disclosure includes variants of the CDRs (i.e. variants ofCDR1, CDR2 and/or CDR3) that can bind to one or more of the sameproteins present in venom recognized by the CDRs (CDR1, CDR2 and/orCDR3) disclosed above.

The present disclosure also includes variants of the isolated V_(H)Hantibody fragments that can bind to one or more of the same proteinspresent in venom recognized by the V_(H)H antibody fragments disclosedabove.

The term “variant” as used herein includes modifications or chemicalequivalents of the amino acid sequences disclosed herein that performsubstantially the same function as the CDRs and/or V_(H)H antibodyfragments disclosed herein in substantially the same way. For example,variants of amino acid sequences disclosed herein include, withoutlimitation, conservative amino acid substitutions. A “conservative aminoacid substitution” as used herein, is one in which one amino acidresidue is replaced with another amino acid residue without abolishingthe binding properties of the CDRs and/or V_(H)H antibody fragments.Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as alanine, isoleucine, valine,leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

A person skilled in the art will appreciate that the present disclosureincludes variants to the amino acid sequences of SEQ ID NOS:5-13, 18 and1-4, including chemical equivalents to the sequences described in thepresent disclosure. Such equivalents include proteins that performsubstantially the same function as the specific proteins disclosedherein in substantially the same way. For example, a functional variantof a CDR will be able to bind to the antigen recognized by the nativeCDR. For example, equivalents include, without limitation, conservativeamino acid substitutions.

In one embodiment, the variant amino acid sequences of the heavy chainCDR1, CDR2 and/or CDR3 have at least 50%, or at least 60%, or at least70%, or at least 80%, or at least 90%, or at least 95% sequence identityto SEQ ID NOS:5-8 and 31; 9-10, 18, 30 and 33; and 11-13, 29 and 32;respectively.

In another embodiment, the variant amino acid sequences of the V_(H)Hantibody fragments have at least 50%, or at least 60%, or at least 70%,or at least 80%, or at least 90%, or at least 95% sequence identity toSEQ ID NOS:1-4.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences. In order todetermine the percentage of identity between two polypeptide sequences,the amino acid sequences of such two sequences are aligned, preferablyusing the Clustal W algorithm (Thompson, J D, Higgins D G, Gibson T J,1994, Nucleic Acids Res. 22 (22): 4673-4680), together with BLOSUM 62scoring matrix (Henikoff S, and Henikoff J. G., 1992, Proc. Natl. Acad.Sci. USA 89: 10915-10919) and a gap opening penalty of 10 and gapextension penalty of 0.1, so that the highest order match is obtainedbetween two sequences wherein at least 50% of the total length of one ofthe sequences is involved in the alignment. Other methods that may beused to align sequences are the alignment method of Needleman and Wunsch(J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv.Appl. Math., 1981, 2: 482) so that the highest order match is obtainedbetween the two sequences and the number of identical amino acids isdetermined between the two sequences. Other methods to calculate thepercentage identity between two amino acid sequences are generallyrecognized and include, for example, those described by Carillo andLipton (SIAM J. Applied Math., 1988, 48:1073) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects. Generally,computer programs will be employed for such calculations. Computerprograms that may be used in this regard include, but are not limitedto, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP,BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990: 215: 403).

In one embodiment, the CDR1, CDR2 and CDR3 and/or V_(H)H antibodyfragments disclosed herein can bind to one or more proteins present insnake venom. In another embodiment, the CDR1, CDR2 and CDR3 and/orV_(H)H antibody fragments disclosed herein can bind to α-cobratoxinpresent in the snake venom. In another embodiment, the CDR1, CDR2 andCDR3 and/or V_(H)H antibody fragments disclosed herein can bind to thesame and/or overlapping epitopes on α-cobratoxin.

The CDRs (CDR1, CDR2 and CDR3) and/or V_(H)H antibody fragmentsdescribed herein may be humanized in order to make them better toleratedfor use in humans. For example, amino acid residues in the frameworkregions may be humanized by replacing them with amino acid residues andthe human framework regions as long as the replacement does not impairthe ability of the CDRs (CDR1, CDR2 and CDR3) and/or V_(H)H antibodyfragments to bind to the toxin (Vincke C, Loris R, Saerens D,Martinez-Rodriguez S, Muyldermans S, Conrath K., “General strategy tohumanize a camelid single-domain antibody and identification of auniversal humanized nanobody scaffold”, J Biol Chem. 2008).

The present disclosure also provides isolated nucleic acid sequencesencoding: the heavy chain complementarity determining region 1 (CDR1)comprising the amino acid sequence: GSISSIYAMG (SEQ ID NO:5); GSTFDDYAIG(SEQ ID NO:6); GDISSFNAMG (SEQ ID NO:7); GSISSFNGMG (SEQ ID NO:8); orG(D/S)ISSFN(A/G)MG) (SEQ ID NO:31); the heavy chain CDR2 comprising theamino acid sequence: VITNGNSPNYADSVKGR (SEQ ID NO:9); FISSGGRSKYTDSVKGR(SEQ ID NO:10); DSVKGR (SEQ ID NO: 18); FISSGGRSKYTDSVK; (SEQ ID NO:30);or VITNGNSPNYADSVKG (SEQ ID NO:33); and the heavy chain CDR3 comprisingthe amino acid sequence: EGVRYGDSWYDGDY (SEQ ID NO:11); GSWSYETGNYYEPSNY(SEQ ID NO: 12); GSVLSYVTGNYYEPSDY (SEQ ID NO:13);GSV(V/L/I)SY(EN)TGNYYEPS(N/D)Y (SEQ ID NO:29); or EGVRYGDSWYDG(D/V)Y(SEQ ID NO:32).

The term “isolated nucleic acid sequences” as used herein refers to anucleic acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors, orother chemicals when chemically synthesized. An isolated nucleic acid isalso substantially free of sequences which naturally flank the nucleicacid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid)from which the nucleic acid is derived. The term “nucleic acid” isintended to include DNA and RNA and can be either double stranded orsingle stranded, and represents the sense or antisense strand. Further,the term “nucleic acid” includes the complementary nucleic acidsequences.

The present disclosure also provides an isolated nucleic acid sequenceencoding the V_(H)H antibody fragments disclosed herein. In oneembodiment, the isolated nucleic acid sequence encodes the V_(H)Hantibody fragment comprising the amino acid sequence shown in SEQ IDNO:1. In another embodiment, the isolated nucleic acid sequence encodesthe V_(H)H antibody fragment comprising the amino acid sequence shown inSEQ ID NO:2. In another embodiment, the isolated nucleic acid sequenceencodes the V_(H)H antibody fragment comprising the amino acid sequenceshown in SEQ ID NO:3. In another embodiment, the isolated nucleic acidsequence encodes the V_(H)H antibody fragment comprising the amino acidsequence shown in SEQ ID NO:4. In a further embodiment, the nucleic acidsequence encoding the V_(H)H antibody fragment comprises the nucleicacid sequence shown in SEQ ID NO:14. In a further embodiment, thenucleic acid sequence encoding the V_(H)H antibody fragment comprisesthe nucleic acid sequence shown in SEQ ID NO:15. In a furtherembodiment, the nucleic acid sequence encoding the V_(H)H antibodyfragment comprises the nucleic acid sequence shown in SEQ ID NO:16. Inan additional embodiment, the nucleic acid sequence encoding the V_(H)Hantibody fragment comprises the nucleic acid sequence shown in SEQ IDNO:17.

The present disclosure also includes variants to the nucleic acidsequences that encode for the CDRs (CDR1, CDR2 and/or CDR3) disclosedherein. For example, the variants include nucleotide sequences thathybridize to the nucleic acid sequences encoding the CDRs (CDR1, CDR2and/or CDR3) of the present disclosure under at least moderatelystringent hybridization conditions.

The present disclosure also includes variants to the nucleic acidsequences that encode for the V_(H)H antibody fragments disclosedherein. For example, the variants include nucleotide sequences thathybridize to the nucleic acid sequences encoding the V_(H)H antibodyfragments of the present disclosure under at least moderately stringenthybridization conditions.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In one embodiment,stringent hybridization conditions are selected. By way of example thefollowing conditions may be employed to achieve stringent hybridization:hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt'ssolution/1.0% SDS at Tm −5° C. based on the above equation, followed bya wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridizationconditions include a washing step in 3×SSC at 42° C. It is understood,however, that equivalent stringencies may be achieved using alternativebuffers, salts and temperatures. Additional guidance regardinghybridization conditions may be found in: Current Protocols in MolecularBiology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2001.

Accordingly, the present disclosure includes isolated nucleic acidsequences encoding variants of the CDRs and/or the V_(H)H antibodyfragments discussed above.

Variant nucleic acid sequences include nucleic acid sequences thathybridize to the nucleic acid sequences encoding the amino acidsequences shown in SEQ ID NOS:5-13, 1-4, 18, and 29-33 and variantsthereof under at least moderately stringent hybridization conditions.

III. Preparation of Proteins

A person skilled in the art will appreciate that the proteins of thepresent disclosure, such as the heavy complementarity determiningregions, the V_(H)H antibody fragments, and the novel isolated proteins,such as V_(H)H antibody fragments isolated from the library describedherein may be prepared in any of several ways, including for example,recombinant methods.

The term “isolated proteins” refers to a protein substantially free ofcellular material and/or culture medium when produced by recombinant DNAtechniques, or obtained from cultured cells or tissue samples, or ofchemical precursors or other chemicals when chemically synthesized.

Accordingly, the nucleic acid molecules of the present disclosure may beincorporated in a known manner into an appropriate expression vectorwhich ensures good expression of the proteins of the present disclosure.Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g. replication defective retroviruses,adenoviruses and adeno-associated viruses), so long as the vector iscompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule of the present disclosure andregulatory sequences selected on the basis of the host cells to be usedfor expression, which is operatively linked to the nucleic acidmolecule. Operatively linked is intended to mean that the nucleic acidis linked to regulatory sequences in a manner which allows expression ofthe nucleic acid.

The present disclosure therefore contemplates a recombinant expressionvector of the present disclosure containing a nucleic acid molecule ofthe present disclosure, or a variant thereof, and the necessaryregulatory sequences for the transcription and translation of theinserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the present disclosure may alsocontain a selectable marker gene which facilitates the selection of hostcells transformed or transfected with a recombinant molecule of thepresent disclosure. Examples of selectable marker genes are genesencoding a protein such as G418 and hygromycin which confer resistanceto certain drugs, β-galactosidase, chloramphenicol acetyltransferase,firefly luciferase, or an immunoglobulin or portion thereof such as theFc portion of an immunoglobulin preferably IgG. Transcription of theselectable marker gene is monitored by changes in the concentration ofthe selectable marker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the present disclosure and inparticular to determine the effect of a mutation on expression andphenotype. It will be appreciated that selectable markers can beintroduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g. a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the present disclosure. Prokaryotic cells can be transformedwith nucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd Edition,Cold Spring Harbor Laboratory Press, 2001), and other laboratorytextbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the present disclosuremay be expressed in yeast cells or mammalian cells. Other suitable hostcells can be found in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1991). In addition,the proteins of the present disclosure may be expressed in prokaryoticcells, such as Escherichia coli (Zhang et al., Science 303(5656): 371-3(2004)). In addition, a Pseudomonas based expression system such asPseudomonas fluorescens can be used (US Patent Application PublicationNo. US 2005/0186666, Schneider, Jane C et al.).

Yeast and fungi host cells suitable for carrying out the presentdisclosure include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY88 (Schultz et al.,Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Protocols for the transformation of yeast and fungi are wellknown to those of ordinary skill in the art (see Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology153:163 (1983), and Cullen et al. (Bio/Technology 5:369 (1987)).

Mammalian cells suitable for carrying out the present disclosureinclude, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g.ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2),293 (ATCC No. 1573) and NS-1 cells. Suitable expression vectors fordirecting expression in mammalian cells generally include a promoter(e.g., derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the present disclosure may beexpressed from plant cells (see Sinkar et al., J. Biosci (Bangalore)11:47-58 (1987), which reviews the use of Agrobacterium rhizogenesvectors; see also Zambryski et al., Genetic Engineering, Principles andMethods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, PlenumPress, New York (1984), which describes the use of expression vectorsfor plant cells, including, among others, PAPS2022, PAPS2023, andPAPS2034).

Insect cells suitable for carrying out the present disclosure includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., Mol.Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow, V. A., andSummers, M. D., Virology 170:31-39 (1989)).

Alternatively, the proteins of the present disclosure may also beexpressed in non-human transgenic animals such as rats, rabbits, sheepand pigs (Hammer et al. Nature 315:680-683 (1985); Palmiter et al.Science 222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

The proteins of the present disclosure may also be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154(1964); Frische et al., J. Pept. Sci. 2(4): 212-22 (1996)) or synthesisin homogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E.Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

Accordingly, the present disclosure provides a recombinant expressionvector comprising the nucleic acid sequences that encode the proteins ofthe present disclosure, such as the heavy chain complementaritydetermining regions, the V_(H)H antibody fragments, and novel isolatedproteins of the present disclosure, such as V_(H)H antibody fragmentsisolated from the library described herein. Further, the presentdisclosure provides a host cell comprising the nucleic acid sequences orrecombinant expression vectors of the present disclosure.

IV. Methods and Uses

The present disclosure includes all methods and uses described herein ofthe CDRs (CDR1, CDR2 and/or CDR3) and/or V_(H)H antibody fragmentsdescribed herein, including their use in therapeutic and diagnosticmethods.

In one embodiment, the present disclosure provides a method of treatinga subject that has been exposed to a venom comprising administering aneffective amount of a CDR and/or V_(H)H antibody fragment to thesubject. The disclosure also includes use of a CDR and/or V_(H)Hantibody fragment to treat a subject that has been exposed to a venom.The disclosure also includes use of a CDR and/or V_(H)H antibodyfragment in the manufacture of a medicament to treat a subject that hasbeen exposed to a venom.

The “subject” can be any member of the animal kingdom, and in oneembodiment is a human.

As used herein “exposed to a venom” means that a subject has beenexposed to and/or has come into contact with a venom as defined herein.Venom exposure could occur, for example, by a bite, sting, or otherwisecontact with a venomous animal (i.e. animal that releases a venom)and/or venomous substance.

In one embodiment, the present disclosure provides a method of treatingenvenomation in a subject comprising administering an effective amountof a CDR and/or V_(H)H antibody fragment to the subject. The disclosurealso includes use of a CDR and/or V_(H)H antibody fragment to treatenvenomation in a subject. The disclosure also includes use of a CDRand/or V_(H)H antibody fragment in the manufacture of a medicament totreat envenomation in a subject.

The term “envenomation” as used herein refers to neurotoxicity and/orneurotoxic effects and/or cytotoxicity that occur after a subject isexposed to a venom. As used herein “neurotoxicity” and/or “neurotoxiceffects” includes, for example, paralytic effects of venom and/orblocking of neuromuscular transmission and/or muscle twitch and/ormuscle flaccid paralysis and/or paralytic effects at neuromuscularjunctions and/or effects of post-synaptic neurotoxins and/or respiratoryfailure. As used herein “cytotoxicity” includes, for example, cell deathand/or cell damage and/or cell necrosis and/or tissue damage and/ortissue necrosis, including for example, local tissue necrosis and/orextensive local tissue necrosis.

“Treating envenomation” or “treat envenomation” as used herein meansthat neurotoxicity, neurotoxic effects and/or cytotoxicity areneutralized. As used herein “neutralized” means counteracting the effectof the venom and/or antidoting the venom, for example, by introducing anantibody and/or other therapeutic molecule that binds to the toxin atits active site and/or or at a protective epitope on the toxin moleculeand/or by binding the toxin and mediating its clearance through theliver.

The present description also includes a method of neutralizing a venomin a subject that has been exposed to the venom comprising administeringan effective amount of a CDR and/or V_(H)H antibody fragment to thesubject. The disclosure also includes use of a CDR and/or V_(H)Hantibody fragment to neutralize a venom in a subject that has beenexposed to the venom. The disclosure also includes use of a CDR and/orV_(H)H antibody fragment in the manufacture of a medicament toneutralize a venom in a subject that has been exposed to the venom.

As used herein “neutralizing a venom” means without limitationcounteracting the effects of a venom and/or antidoting the effects of avenom, for example, reducing the cytotoxicity and/or neurotoxicityand/or neurotoxic effects of the venom, which includes for example: areduction in the paralytic effects of venom and/or a reduction inparalysis and/or a reduction in muscle twitch and/or a reduction in theblocking of neuromuscular transmission and/or a reduction in muscleflaccid paralysis and/or a reduction in the paralytic effects atneuromuscular junctions and/or a reduction of effects caused bypost-synaptic neurotoxins and/or a reduction in respiratory failureand/or a reduction in cell death and/or cell damage and/or cell necrosisand/or tissue damage and/or tissue necrosis, including for example,local tissue necrosis and/or extensive local tissue necrosis.

The CDR and/or V_(H)H antibody fragment is one that binds to a proteinand/or toxin present in the venom to which the subject has been exposed.In one embodiment, in the methods and used described above, the subjectis given several different CDRs and/or V_(H)H antibody fragmentsdisclosed herein, each one specific for a different protein and/or toxinpresent in the venom.

In another embodiment, in the methods and used described above, the CDRsand/or V_(H)H antibody fragments bind to one or more proteins and/ortoxins present in a venom. In another embodiment, in the methods andused described above, the CDRs and/or V_(H)H antibody fragments bind toone or more proteins and/or toxins present in snake venom. In yetanother embodiment, in the methods and used described above, the CDRsand/or V_(H)H antibody fragments bind to α-cobratoxin present in thesnake venom. In another embodiment, in the methods and used describedabove, the CDRs and/or V_(H)H antibody fragments bind to the same and/oroverlapping epitopes on α-cobratoxin.

V. Pharmaceutical Compositions

The present disclosure also includes a pharmaceutical compositioncomprising an effective amount of CDRs and/or V_(H)H antibodyfragment(s) of the disclosure in admixture with a suitable diluent orcarrier.

Administration of an “effective amount” of the pharmaceuticalcompositions is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, atherapeutically active amount of a substance may vary according tofactors such as the disease state, age, sex, and weight of the subject,and the ability of the CDR and/or V_(H)H antibody fragment to elicit adesired response in the subject. Dosage regime may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The form of administration or use will depend on the nature and locationof the venom. Suitable forms of administration include systemic(subcutaneous, intravenous, intramuscular), oral administration,inhalation, transdermal administration, topical application (such astopical cream or ointment, etc.) or by other methods known in the art.

Accordingly, the present disclosure provides a pharmaceuticalcomposition for treating a subject exposed to a venom and/or treatingenvenomation in a subject and/or neutralizing a venom in a subject thathas been exposed to the venom comprising one or more of the CDRs and/orV_(H)H antibody fragments in admixture with a suitable diluent orcarrier.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, 20^(th) ed., Mack Publishing Company, Easton, Pa., USA, 2000).On this basis, the compositions include, albeit not exclusively,solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. Immunoconjugate may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

VI. Kits

The present disclosure also provides a kit comprising an effectiveamount of V_(H)H antibody fragment(s) disclosed herein, together withinstructions for the use thereof. In one embodiment, the use thereofincludes treating a subject exposed to a venom and/or treatingenvenomation in a subject and/or neutralizing a venom in a subject thathas been exposed to the venom. The kit can also include ancillaryagents. For example, the kits can include instruments for injecting theV_(H)H antibody fragment(s) disclosed herein into a subject, such as asyringe; vessels for storing or transporting the V_(H)H antibodyfragment(s) disclosed herein and/or a pharmaceutically suitable diluentor carrier.

The following non-limiting examples are illustrative of the presentdisclosure:

Example 1 Materials and Methods Immunization

A llama (Lama glama) was injected subcutaneously with increasing dosesof crude N. kaouthia venom (Latoxan, France) every three weeks for a15-week period.

The immunogen was administered subcutaneously at three differentlocations (0.25 mL/site) (i.e., one site near the neck and two in thehind quarters).

ca. 75 ml of blood was collected one week after each immunization forantibody specific titrations.

After collection of pre-immune sera (day −7), a one-year-old male llama(Lama glama) was immunized on days 0, 14, 35, 56, 75 and 103 withincreasing amounts (i.e. 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg,respectively) of crude N. kaouthia venom. The venom [dissolved inphosphate buffered saline (PBS), pH 7.4] was emulsified in an equalvolume of TiterMax™ Classic Adjuvant (Sigma-Aldrich, Oakville, ON,Canada) for the first three immunizations, and emulsified with an equalvolume of Freund's Incomplete Adjuvant (Sigma-Aldrich, Oakville, ON,Canada) for subsequent immunization. The immunogen was administeredsubcutaneously at three different locations (0.25 mL/site) (i.e., onesite near the neck and two in the hind quarters), which was similar toscheme described by Chotwiwatthanakun et al. (2001). Immune bleeds (˜100mL) were withdrawn from the jugular vein on days 21, 42, 63, 82 and 110.Serum was collected by centrifuging at 2,700×g for 10 min, aliquoted andstored at −20° C. until required for use. Whole llama blood was alsocollected, at days 82 and 110, in a glass vacuum bottle containing 1-2mg/mL of EDTA (dipotassium salt) to prevent coagulation. Thereafter,peripheral blood leukocytes were recovered from 1.5 mL aliquots of thiswhole blood, lyzed as described in the QIAamp RNA Blood Mini Kit(Qiagen), and stored at −80° C. until required for RNA isolation.

Immune Response

Sera from days 42, 63, 82 and 110 were fractionated into HCAb and cIgGby Protein G and Protein A chromatography using a gradient pH elution(Hamers-Casterman et al., 1993).

HCAb immune response to α-Cbtx was monitored by enzyme-linkedimmunosorbant assay (ELISA).

The polyclonal immune responses against N. kaouthia venom components andα-Cbtx were monitored over the course of the llama immunization byindirect enzyme-linked immunosorbent assay (ELISA). Wells of aReacti-Bind™ maleic anhydride-activated polystyrene microtitre plate(Pierce Biotechnology, Rockford, Ill.) were coated with 1 ug/mL of crudeN. kaouthia venom or with 2.5 ug/mL of α-Cbtx (100 uL/well; PBS, pH 7.4)overnight (o/n) at 4° C. Negative background control wells were notcoated with the antigen (PBS only). Wells were washed 3× with 200 uL ofPBS (pH 7.4) to remove unbound antigen and then blocked o/n with 300 uLof 4% MPBS [4% (w/v) milk powder in PBS, pH 7.4]. Llama polyclonal serumfrom days −7, 21, 63, 110 were diluted by a serial two-fold dilutionstarting with a 1:50 dilution, added to the wells (100 uL/well), andincubated with gentle shaking at room temperature (RT). After 1.5 hrincubation, serum samples were removed and the wells were washed 3× with200 uL of PBS-T (PBS plus 0.05% (v/v) Tween-20). Goat anti-llamaIgG-heavy and light-chain conjugated to HRP (horse radish peroxidase)(Bethyl, lab Inc, 6 Montgomery, Calif.) diluted 1:2000 in 4% MPBS wasadded to the wells (100 uL/well) and incubated for 1 hr at RT withgentle shaking. Wells were washed 3× with 200 uL of PBS-T, and thendeveloped with 100 μL/well of TMB substrate (3,3′,5,5′-tetramethylbenzidine; Pierce, Rockford, Ill.). After 10 min, the reactions wereneutralized with 1.5 M H₂SO₄. (100 μL/well) and the level of binding wasdetermined spectrophotometrically at 450 nm.

After detecting a polyclonal immune response, a specific HCAb immuneresponse against α-Cbtx was also determined. Llama sera werefractionated into HCAbs and convIgGs using protein G chromatography asdescribed by Hamers-Casterman et al. (1993) with minor modifications.Four mL of sera from days 21 and 110 post-immunization and day −7(pre-immune negative control) were dialyzed o/n against PBS (pH 7.4)using dialysis tubing with a 12-14 kDa MW cutoff. Dialyzed sera werediluted 10-fold in PBS (final volume 40 mL) and loaded onto a 5-mLprotein G column (Hitrap, Pharmacia, Upsala, Sweden) using an AKTA FPLCsystem (GE Healthcare Bio-sciences AB, Uppsala, Sweden). After washingthe column with PBS (pH 7.4), the HCAb fraction G1 was first elutedusing 0.1M Citrate buffer (pH 3.5) and then the convIgG fraction G2 waseluted using 0.1M Glycine-HCl (pH 2.3). After elution, fractions wereimmediately neutralized with 1M Tris-HCl (pH 8.8), dialyzed o/n againstPBS (pH 7.4), filtered through 0.22 μm and stored at 4° C. The purity ofconvIgG and HCAb fractions were determined by standard SDS-PAGE andWestern blotting. Protein concentrations were spectrophotometricallymeasured at wavelength of 280 nm.

The HCAb immune response against α-Cbtx was assessed by indirect ELISA,as above, with the following exception: instead of using polyclonalserum, HCAb fractions G1 were titrated against microtitreplate-immobilized α-Cbtx (10 ug/mL).

V_(H)H Library Construction

To construct a V_(H)H library, total RNA was extracted from leukocytesof the 5^(th) bleed (Day-110).

After detecting a HCAb-positive immune response against α-Cbtx fromfractionated sera, the V_(H)H library was constructed following themethods of Ghahroudi et al. (1997) with minor modifications. Total RNAwas extracted from one aliquot (1.5 mL) of lyzed leukocytes (as above)from day 110 using the QIAamp RNA Blood Mini™ kit (Quiagen).Subsequently, this RNA (2 μg/8 uL) was used as template for thesynthesis of the first strand cDNA using the First-Strand cDNA synthesiskit (Amersham Biosciences, Buckinghamshire, UK) and random hexamers[pd(N)6] as primers. Two cDNA synthesis reactions were carried out andpooled. The cDNA concentration was not quantified.

V_(H)H gene repertoire was amplified, cloned into pMED1 phagemid vector(Arbabi-Ghahroudi et al., 1997) and transferred into TG1 E. coli.

The first round of PCR reactions used framework-1 specific sense primers(MJ1.2.3 Back; Table 1) and CH2-specific anti-sense primers (CH2 andCH2B3; Table 1), thereby amplifying the V_(H)H-C_(H)2 andV_(H)-C_(H)1-C_(H)2 regions of HCAb and convIgG genes, respectively. Tooptimize the amplification of the V_(H)H gene segment, small-scale testPCR reactions were first carried out with various amounts of cDNA (0.5-3uL) and MgCl₂ (0.5-3.0 mM). The optimal conditions for the amplificationusing the CH2 primer used 3.0 uL of cDNA and 1.5 mM MgCl₂. The PCRcycling parameters were as followed: 94° C. for 5 min (Taq hot start);30 cycles of: 94° C. for 45 sec, 57° C. for 45 sec, 72° C. for 1.5 min;72° C. for 7 min; 4° C. ∞.

The optimal conditions for the amplifications using the CH2B3 primerused 3.0 uL of cDNA and 0.5 mM MgCl₂. The PCR cycling parameters were asfollowed: 94° C. 5 min (Taq hot start); 6 cycles of: 94° C. for 45 sec,57° C. for 45 sec, 54° C. for 45 sec, 72° C. for 1.5 min; 24 cycles of:94° C. for 45 sec, 57° C. for 45 sec, 72° C. for 1.5 min; 72° C. for 7min; 4° C. ∞.

Ten 50-μL PCR reactions of both primer sets (CH2 and CH2B3) were carriedout with 5 pmol of the respective primers, 0.25 mM dNTPs and 2.5 unitsof Taq DNA polymerase (Hoffmann-La Roche Ltd.; Mississauga, ON). The 10respective PCR products were pooled and electrophoresed on a 2% agarosegel to separate the V_(H)H-C_(H)2 (˜600 bp) band from theV_(H)-C_(H)1-C_(H)2 (˜900 bp) fragment. The V_(H)H-C_(H)2 (˜600 bp) bandwas excised and purified using the Qiagen QIAquick Gel Extraction kit(QIAGEN Inc., Mississauga, ON, Canada).

Subsequently, the amplified V_(H)H-C_(H)2/-C_(H)2B3 products were usedas template DNA for nested PCRs using primers specific for theextremities of framework-1 (MJ7; sense) and framework-4 (MJ8;anti-sense; Table 1), resulting in an amplified V_(H)H fragment withoutthe C_(H)2 gene segment. These primers also introduced Sfi I restrictionsites (5′-GGCCNNNN̂NGGCC-3′ (SEQ ID NO:28); See Table 1 underlinedsequences) at the 5′ and 3′ end of the V_(H)H sequence. To optimize theV_(H)H gene amplification, small-scale test PCR reactions were performedwith various amounts of V_(H)H-C_(H)2 or V_(H)H-C_(H)2B3 as template DNA(1-3 uL) and MgCl₂ (0.25-1.5 mM), and with different annealingtemperatures. The optimal conditions for the amplifications of theV_(H)H gene segment used 5 ng of V_(H)H-C_(H)2 or 10 ng ofV_(H)H-C_(H)2B3 amplicons as template DNA and 1.0 mM MgSO₄. Twenty 50-μLPCR reactions for both sets were done with 3.5 μmol of MJ7 and MJ8primers, 0.25 mM dNTPs and 2.5 units of Taq DNA polymerase. An aliquot(˜3 uL) from each PCR product was run on a 1% agarose gel to confirm theamplified product was of the correct size for a V_(H)H (˜450 bp). Allthe PCR products were pooled and purified using MinElute spin columns(MinElute PCR Purification Kit; QIAGEN Inc., Mississauga, ON, Canada).

The V_(H)H gene repertoire (10 ug) was digested with Sfi I (New EnglandBiolabs, Ipswich, USA) at 50° C. for 24 hr. After digestion, a smallaliquot was analyzed on a 1% agarose gel to confirm it was the propersize for a V_(H)H fragment. The V_(H)H-Sfi I insert was purified usingMinElute spin columns.

Twenty μg of pMED1 phagemid vector (Ghahroudi et al., 1997) weredigested with Sfi I for 24 hr at 50° C., purified using QIAquick PCRpurification kit, and double-digested with Pst I (Roche) and Xho I(Roche) for 5 hr at 37° C. The digested vector was purified with theQIAquick PCR purification kit and concentrated by standard ethanol DNAprecipitation. Several test ligations/transformations were done tooptimize the V_(H)H cloning (% insert) and transformation efficiencies.After optimization, twenty-seven small-scale ligation reactions wereserially done with 200 ng vector, 75 ng of V_(H)H-Sfi I insert, 1 uL ofT4 DNA ligase (Promega, Madison, Wis.), 1 uL of Buffer at 16° C. for 16hr. The ligation material was pooled, purified using spin columnsprovided in the QIAquick PCR purification kit, and eluted in a totalvolume of 200 uL ddH₂O.

Four uL of the pMED1-V_(H)H ligated product were transformed into 50 uLof prepared electrocompentent E. coli TG1 at 1200 V, 25 pF and 200Ωusing a Gene Pulser XceII™ electroporator (Bio-Rad Laboratories,Mississauga, ON, Canada) and 0.1-cm electroporation cuvettes (Bio-RadLaboratories, Mississauga, ON, Canada). Immediately aftertransformation, the cells were transferred to 1 mL of pre-warm SOCmedium, and incubated with shaking for 1 hr at 37° C. A total of 50transformations were done. After the 1 hr of incubation, the fifty 1-mLcultures were pooled. To determine the size of the library, a smallaliquot (10 uL) of transformed cells was serially diluted (10⁻⁴, 10⁻⁵and 10⁻⁶) and plated onto 2×YT agar plates containing ampicillin (100μg/mL) and 1% (w/v) glucose (2×YT/Amp/1% glucose). The culture wascentrifuged at 3,000 g for 20 min and resuspended in 500 mL of2×YT/Amp/2% glucose. The library was amplified o/n at 37° C. at 220 rpm.The next morning, the library was centrifuged as described above andresuspended in 100 mL of 2×YT/Amp/2% glucose with glycerol (30% finalconcentration). The amplified library was aliquoted (˜5.0×10⁹ bacterialcells/aliquot; 3.5 mL), and stored at −80° C. until required for use.

Selection of α-Cbtx V_(H)H Binders

The V_(H)H phage-displayed library was panned three rounds againstimmobilized α-Cbtx.

For panning round 1, one well of a Reacti-Bind™ maleicanhydride-activated polystyrene microtitre plate (Pierce Biotechnology,Rockford, Ill.) was coated with sterile PBS (pH 7.4; 100 uL) and asecond well was coated with 40 ug of α-Cbtx (diluted in sterile PBS, pH7.4; 100 uL). For panning rounds 2 and 3, the coating concentration ofα-Cbtx was decreased to 20 and 5 ug, respectively. After o/n incubatedat 4° C., wells were washed 3× with 200 uL PBS and blocked with 300 uLof 4% MPBS at 37° C. for 2 hours. During this time, 100 uL of amplifiedphage and 100 uL of 8% MPBS (1:1 phage:blocking agent ratio) werecombined in a 0.5 mL tube and pre-incubated with rotation for 1 hr atRT. After the blocking incubation was complete, the wells were washed 5×with PBS (300 uL). For subtractive panning of plastic binders, 100 uL ofthe pre-incubated phage were first incubated in the PBS coated well for1 hr at 37° C. After incubation, the content of the well was transferredto the α-Cbtx coated well and incubated for 2 hr at 37° C. Unboundphages were removed by washing 5, 8 and 12× with PBST (200 uL) forpanning rounds 1, 2 and 3, respectively, and then washed 2× with PBS(200 uL). To elute bound phages, 200 uL of 100 mM triethylamine (TEA)was added to the well and incubated at RT for 10 min. For the last 2 minof this incubation, the content of the well was stirred by pipetting upand down several times. Eluted phages were transferred to amicrocentrifuge tube and vortexed with 400 uL of 1 M Tris-HCl (pH 7.4)to neutralize the TEA. Eluted phage (600 uL) were used to infect 1.4 mLof exponentially growing E. coli TG1 without shaking for 30 min at 37°C. After infection, a 10 uL aliquot of infected E. coli was used to makea serial dilution (from 10⁻² to 10⁻⁶) to determine the phage titre(output). The remaining culture was spread on a 2×YT/Carb/1% glucoseagar plate and incubated o/n at 32° C. The next morning, the cells wereloosened from the plate using a plastic loop and 2 mL of 2×YT/Carb/15%glycerol. A 100 uL aliquot of these cells were inoculated into 50 mL of2×YT/Carb/1% glucose for the production of phage for the next round ofpanning. (The remaining cells were stored at −80° C.). When theabsorbance reading reached 0.4, 10¹¹ pfu of M13KO7 helper phage wasadded to 10 mL of the E. coli culture. The growing of the culture andphage harvesting were performed as previously described in preparationfor the next round of panning.

After 3 rounds of panning, 46 random clones were screened by monoclonalphage ELISA. Clones positive for binding to α-Cbtx were sequenced.

A total of 46 colonies (from the titre plate) from the third round ofpanning were screened for α-Cbtx binding by monoclonal phage ELISA.Colonies were grown in a 96-well culture plate (Corning IncorporatedLife Sciences, Acton, Mass.) containing 100 uL/well of 2×YT supplementedwith ampicillin (100 ug/mL) and 1% glucose. After incubation at 37° C.for 16 hr with shaking (220 rpm), 2 uL of each o/n culture wastransferred into 200 uL of 2×YT supplemented with ampicillin and 1%glucose. Culture plates were incubated at 37° C. for 2 hr with shaking.After incubation, each culture was infected with 10¹⁰ helper phage(M13KO7) and incubated at 37° C. for 15 min without shaking and then for1 hr with shaking (250 rpm). The culture plate was centrifuged at 1800rpm for 10 min at 4° C., and the supernatant was carefully removed anddiscarded. Cell pellets were resuspended in 200 uL of 2×YT containingampicillin (100 ug/mL) and kanamycin (50 ug/mL), and grown o/n withshaking (250 rpm) at 30° C. After centrifugation of the plate for 30 minas described above, 50 uL supernatant (which contain theV_(H)H-displayed phages) were collected and used for monoclonal phageELISA.

Reacti-Bind™ maleic anhydride-activated polystyrene microtitre plates(Pierce Biotechnology, Rockford, Ill.) were coated with α-Cbtx (1μg/mL), blocked with SuperBlock, and washed as described previously.Phage supernatant (50 μL) and SuperBlock (50 μL) were added to wells andincubated at 37° C. for 2 hr. Wells were washed and bound phage weredetected as described above.

Clones with absorbance (450 nm) readings greater than 0.3 backgroundwere sequenced using the universal M13RP primer at the LaboratoryDivision Services (University of Guelph).

Soluble V_(H)H Protein Expression

Four unique anti-α-Cbtx V_(H)H clones (C2, C19, C20 and C43) wereselected for soluble expression in E. coli HB2151 and purified fromperiplasm/cytoplasm extracts by IMAC using “HiTrap™ Chelating HP”column.

Preliminary expression and purification results showed that C2 and C43clones expressed at low levels in pMED1 phagemid vector. Therefore,these V_(H)H coding sequences were subcloned into the Sfi I restrictionsites of the expression vector pMED2 (kindly provided by Dr. MehdiArbabi-Ghahroudi). Proteins expressed from both pMED1 and pMED2 vectorscontain a His₆ tag for purification.

For soluble expression of V_(H)Hs, purified recombinant C2-pMED2,C19-pMED1, C20-pMED1 and C43-pMED2 constructs were electroporated intoE. coli strain HB2151, a non-suppressor strain. Single colonies werepicked and transferred into 5 mL of 2×YT starter culture supplementedwith 75 ug/mL carbicillin and 1% (w/v) glucose. Cultures were grown o/nat 37° C. while shaking at 220 rpm. For large-scale expression, 1 mL ofstarter culture was transferred into 1 L of 2×YT medium supplemented asdescribed above and grown at 37° C. while shaking at 220 rpm until theOD₆₀₀ reached 0.6-0.7. The cell pellets were collected by centrifugingat 3,000 g and resuspended in 1 L of 2×YT medium supplemented with 0.1%(w/v) glucose and 75 μg/mL carbicillin. To induce soluble V_(H)Hexpression, IPTG (1 mM final conc.) was added to the cultures. Cultureswere grown at 26° C. for 24 hr while shaking at 220 rpm. The inducedcultures were centrifuged at 8,000 g for 15 min at 4° C. The harvestedcells were resuspended in 100 mL of ice-cold lysis buffer (50 mMTris-HCl at pH 8.0, 25 mM NaCl, 2 mM EDTA), and stored at −20° C. untilneeded for protein extraction.

V_(H)Hs were purified from the periplasmic fractions of E. coli. Theinduced cell pellets that were stored in lysis buffer (100 mL) weretaken out of −20° C. freezer and 1 mL of 100 mM protease inhibitorphenylmethylsulphonyl fluoride (PMSF; 1 mM final conc.; Sigma-Aldrich,Oakville, ON) and 200 μL of 1M dithiothreitol (DTT; 2 mM final conc.;Bioshop, Burlington, ON) were immediately added. The frozen suspensionwas thawed at RT with occasional shaking. To lyse the cells, 5 mL offreshly prepared lysozyme (100 μg/ml final conc. from 3 mg/ml aqueoussolution; Roche, Indianapolis, Ind.) were added to the thawed cells. Thesuspension was incubated at RT for 30-50 min with occasional shaking.When the suspension became viscous 300 μL of DNase I (Sigma,Sigma-Aldrich, Oakville, ON; 15 units/μl stock in 1 M MgCl₂) was addedand the lysate was incubated at RT until the suspension became watery(ca. 20-30 min). The lysate was centrifuged at 12,000 rpm for 20 min at4° C. to separate the soluble and insoluble fractions. Centrifugationwas repeated with the soluble fraction until it became clear. Thesoluble fraction which contained soluble V_(H)Hs were dialyzed o/nagainst PBS (pH 7.4) containing 1 mM EDTA. Samples were filtered througha 0.22 μm sterile filter (Millipore, Nepean, ON). V_(H)Hs were purifiedby standard immobilized metal affinity chromatography (IMAC) using a 5mL HisTrap™ HP nickel affinity column (GE Healthcare Bio-sciences AB,Uppsala, Sweden).

Western blots were done to confirm expressions and purifications. Thetagged V_(H)Hs were detected using anti-penta His monoclonal antibody(Qiagen, Mississauga, ON) diluted 5000 fold, and goat anti-mouse mAbconjugated to alkaline phosphatase (GAM-AP) diluted 5000 fold. Membraneswere washed and developed with alkaline phosphatase substrate (1-StepNBT/BCIP; Pierce Biotechnology, Rockford, Ill.). Fractions containingV_(H)H were dialyzed o/n against SPR analysis buffer (10 mM HEPES, 150mM NaCl, 3 mM EDTA; HBS-E) with a 3500 MW cutoff. Absorbancies weremeasured at 280 nm (A₂₈₀), and V_(H)H concentrations were estimatedusing an extinction coefficient based on the predicted amino acidsequence of each V_(H)Hs computated athttp://ca.expasy.org/tools/protparam.html. The extinction coefficientsfor the purified clones were: C2, 2.089 (mg/mL); C19, 1.742 (mg/mL);C20, 1.744 (mg/mL); C43, 2.090 (mg/mL). The purified V_(H)Hs were storedat 4° C.

Affinity Measurements

Kinetic studies were performed for anti-α-Cbtx V_(H)H C2, C19, C20 andC43 using surface plasmon resonance (SPR).

Binding kinetic experiments were performed by SPR using a Biacore 3000instrument (Biacore Inc., Piscataway, N.J.). Approximately 137 resonanceunits (RUs) of α-Cbtx were immobilized using standard amine couplingonto a research grade CM5 sensor chip (Biacore Inc.) in 10 mM acetatebuffer. Prior to SPR analysis, V_(H)H samples purified from periplasmicfractions of E. coli (above) were subjected to Superdex 75 gelfiltration chromatography (GE Healthcare) to isolate monomers fromaggregates. V_(H)H monomers were passed over the sensor chip coated inHBS-EP running buffer [10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA,0.005% surfactant P-20 (GE Healthcare)]. The four V_(H)Hs C2, C19, C20and C43 were injected at concentrations ranging from 2.5 to 30 nM, 6.25to 150 nM, 1 to 32 nM and 5 to 160 nM, respectively. All experimentswere conducted at RT at a flow rate of 40 μL/min.

Epitope Competition

Epitope competition was performed using SPR to determine if thedifferent V_(H)H clones target different, non-overlapping epitopes onα-Cbtx.

SPR analysis was used to determine if the α-Cbtx binders target the sameor different epitopes. α-Cbtx was immobilized to the sensor chip asdescribed above and then saturated with C19 or C43. After saturation,the ability of the second α-Cbtx binder to simultaneously bind thecomplex was monitored. To ensure the surface capacity (Rmax) wasreached, α-Cbtx binders C2, C19, C20 and C43, were injected at 1 μM, 600nM, 500 nM and 2 uM, respectively, as determined by the kinetic analysisresults (Refer to “Affinity Measurements” section above). All assayswere done at RT with a flow rate of 40 μL/min in HBS-EP running buffer.The RU signal created after the injection of the second α-Cbtx binder isproportional to the binding concentration of this V_(H)H.

Immobilized α-Cbtx was first saturated with either V_(H)H C19 or C43.After saturation was reached, another V_(H)H clone was applied andchange in Response Difference was monitored. Increase in RD wouldindicate binding of second V_(H)H to a different epitope; no difference,binding to same epitope.

Comparison of Amino Acid Sequences for V_(H)H C2, C19, C20 and C43

Amino acid sequences for V_(H)H C2, C19, C20 and C43 were compared usingthe Basic Local Alignment Search Tool (BLAST) on the National Center forBiotechnology Information website<http://www.ncbi.nlm.nih.gov/BLAST/Blast.cqi?CMD=Web&PAGE_TYPE=BlastHome>.

A consensus sequence for the amino acid sequences of V_(H)H C2, C19, C20and C43 was determined using the ClustalW tool at<http://www.ebi.ac.uk/Tools/clustalw2/index.html>.

In Vitro α-Cbtx Neutralization Assay

A functional assay to assess the ability of V_(H)H C2 to neutralize theparalytic effects of α-Cbtx was investigated.

Methodology involved measurement of the force of contraction of a ratdiaphragm muscle preparation stimulated at the phrenic nerve, in thepresence of α-Cbtx with or without V_(H)H C2.

The diaphragm-phrenic nerve preparation is a widely used nerve-musclepreparation and was used to investigate the neutralization efficienciesof isolated V_(H)H clones to α-Cbtx at neuromuscular junctions. Theassay was conducted in a manner similar to that described by Bulbring(1946). Briefly, the left hemi-diaphragm with the attached phrenic nervewas carefully excised from male Sprague Dawley Rats (175-250 g), andmounted on a special tissue holder immersed in a 50-mL Schuler organbath containing Krebs-Henseleit solution. The incubation bath wasmaintained at 37° C. and fed constantly with 95% O₂-5% CO₂ by bubblingthe gas through the solution. The preparation was attached via a threadsuture to a Harvard isometric transducer for recording of contractionson a ®Biopac Systems MP150. The diaphragm contracted in response to“direct” stimulation using a set of parallel electrodes, which alsoserve to anchor the diaphragm, or to “indirect” stimulation via a secondset of electrodes which stimulates the phrenic nerve to releaseneurotransmitter.

The phrenic nerve was continuously stimulated with supramaximal squarewave pulses (0.25 ms) at a frequency of 0.1 Hz., followed by three 3-secperiods, at 30 sec intervals, at frequencies of 25, 50 and 100 Hz., toevoke twitch and tetanic reponses respectively, using a Grass® S88stimulator. This protocol was repeated every 15 min in the presence orabsence of neurotoxin with appropriate changes of bath fluid. In thiscase the tetanic response to 100 Hz was measured and expressed asarea/volt-sec by the Biopac software program.

Purified V_(H)H C2 (100 nM) was pre-incubated with α-Cbtx (50 nM) for 60min at RT prior to adding to the tissue bath. The assay was alsoperformed with 50 nM of α-Cbtx (without V_(H)H) and served as a negativecontrol. As a positive control, the tissue preparation was stimulatedwithout the presence of α-Cbtx and V_(H)H C2.

Results Immune Response

HCAb and cIgG fractions showed specific binding to both crude N.kaouthia venom and purified α-Cbtx (data not shown).

V_(H)H Library Construction

A V_(H)H phage-displayed library was constructed from the 5^(th) bleed.

A library of 4.2×10⁹ clones with 84% V_(H)H-insert ratio wasconstructed.

Sequencing showed 100% diversity among 25 random clones.

Panning, Phage ELISA, and Sequence Results

Polyclonal phage ELISA using eluted phage from each round of panningshowed a saturated signal with the 3^(rd) round of panning (data notshown).

Out of 46 random phage clones, 26 clones were positive for binding toα-cobratoxin by monoclonal phage ELISA.

Sequence analysis showed that 25 of these had unique sequences.

Analysis of the coding sequences and predicted amino acid compositionsrevealed that the 3^(rd) round of panning generated several uniqueα-Cbtx binders. Of all the clones sequenced, only two clones, C15 andC46, shared 100% identity. All other clones were unique with at leastone different amino acid substitution. Many clones shared high identityas revealed by a multiple sequence alignment (MSA) of the predictedamino acid composition (FIG. 6). Moreover, based on the CDR homology andCDR3 length, two distinct groups of α-Cbtx binders are apparent,hereafter named Cluster I and Cluster II (see FIG. 6).

The nine clones (C33, C46, C15, C7, C13, C19, C34, C31, C20) that formCluster I are characterized with a CDR3 length of 17 amino acid residueswith the following consensus motif: GSV(V/L/I)SY(E/V)TGNYYEPS(N/D)Y (SEQID NO:29). All these binders have identical CDR2 regions(FISSGGRSKYTDSVK; (SEQ ID NO:30)), except for C31, which has a “T” atposition 3 of SEQ ID NO:30, and a well conserved CDR1 (consensus motif:G(D/S)ISSFN(A/G)MG) (SEQ ID NO:31).

In contrast to Cluster I, clones that form Cluster II (C2, C29, C43, andC42) have a shorter CDR3 region with 14 amino acid residues, which hasthe following highly conserved consensus motif: EGVRYGDSWYDG(D/V)Y (SEQID NO:32). The clones among Cluster II share an identical CDR2 with thesequence VITNGNSPNYADSVKG (SEQ ID NO:33). Furthermore, all the clonesfrom this group have a conserved CDR1 with the sequence GSISSIYAMG (SEQID NO:5), except C43 which has a unique CDR1.

Analyses of the predicted amino acid sequences suggest two major classesof antibodies (see Table 2. CDR3). Four α-Cbtx V_(H)H clones with highabsorbance values as determined by ELISA (data not shown) were selectedfor further characterization; two from Cluster I (C19 and C20) and twofrom Cluster II (C2 and C43). As shown in Table 2, C2 and C43 differ bynine amino acid residues which are located in FR1 and CDR1. C19 and C20differ by 10 amino acid residues which are located in different regions.

Full sequences for the CDR1, CDR2 and CDR3 are also shown in Table 2.Nucleotide sequences and predicted amino acid sequences foranti-α-cobratoxin V_(H)H C2, C19, C20 and C43 clones are shown in Table5.

Comparison of Amino Acid Sequences for V_(H)H C2, C19, C20 and C43

The amino acid sequences of V_(H)H C2, C19, C20 and C43 was compared todetermine the homology among the four sequences. The % identity and %similarity for V_(H)H C2, C19, C20 and C43 is shown in Table 6.

An overall comparison of the amino acid sequences for V_(H)H C2, C19,C20 and C30 was performed using Clustal W, and a consensus sequence forthese four V_(H)Hs was determined. The consensus sequence showsconserved regions for V_(H)H C2, C19, C20 and C30, and is shown in Table7.

Affinity Measurements

Surface plasmon resonance showed K_(D) values ranged between 0.4-25 nM(FIG. 3 and Table 3).

Epitope Competition

It appears that the different V_(H)H clones target a region ofoverlapping or close together epitopes (FIG. 4).

In Vitro α-Cbtx Neutralization Assay

C2 neutralizes the paralytic effects of α-Cbtx at neuromuscularjunctions (FIG. 5).

Discussion

A positive HCAb immune response against α-Cbtx was generated using crudevenom.

The V_(H)H library had a diversity 100% with a library size of 4.2×10⁹clones.

Four V_(H)H clones were characterized and had K_(D)s for α-Cbtx rangingfrom 0.4-25 nM.

These clones had a much higher affinity than those (2 to 3 uM) selectedfrom a naïve phage-displayed V_(H)H library (Stewart et al., 2007).

In vitro muscle-twitch assay showed that anti-α-Cbtx V_(H)H C2neutralizes the paralytic effects of alpha-cobratoxin.

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Nucleotide sequence of primers used for theconstruction of the V_(H)H phage-displayed library. Primer nameNucleotide sequence MJ1 Back 5′-GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA-3′ (SEQ ID NO: 19) MJ2 Back5′-GCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGT CTGGGGGA-3′ (SEQ ID NO: 20)MJ3 Back 5′-GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGG TGGAGTCT-3′(SEQ ID NO: 21) CH2 5′-CGCCATCAAGGTACCAGTTGA-3′ (SEQ ID NO: 22) CH2B35′-GGGGTACCTGTCATCCACGGACCAGCTGA-3′ (SEQ ID NO: 23) MJ75′-CATGTGTAGACTCGCGGCCCAGCCGGCCATGGCC-3′ (SEQ ID NO: 24) MJ85′-CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGAC GGTGACCTGG-3′ (SEQ ID NO: 25)PN2 5′-CCCTCATAGTTAAGCGTAACGATCT-3′ (SEQ ID NO: 26) M13RP5′-CAGGAAACAGCTATGAC-3′ (SEQ ID NO: 27)

TABLE 2 Amino acid alignment of four selected anti-Cbtx V_(H)H clones.The dots in the sequence represent 100% identity with V_(H)H #2 andthe dashes represent no amino acid. Clone FR1 CDR1 FR2 CDR2 #2QVKLEESGGGLVLPGGSLRLSCAAS GSISSIYAMG WYRQAPGKQREVVA VITNGNSPNYADSVKGR#43 R...........QA.........V. ..TFDD..I. ............................... #19 ...........AQA........IG. .DI..FN.......V......L.. F.SS.GRSK.T...... #20 ............Q.........VG...I..FNG.. ....V......L.. F.SS.GRSK.T...... SEQ ID Clone FR3 CDR3 FR4NO: #2 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCNV EGV---RYGDSWYDGDY WGQGTQVTVSSG 1#43 ................................ ...---........... ............ 2#19 .....G...........ID............A GS.VSYET.NYYEPSN. ............ 3#20 .....G....S......IN............. GS.LSYVT.NYYEPSD. ............ 4SEQ ID SEQ ID SEQ ID CDR1 NO: CDR2 NO: CDR3 NO: C#2 GSISSIYAMG 5VITNGNSPNYADSVKGR 9 EGVRYGDSWYDGDY 11 C#43 GSTFDDYAIG 6VITNGNSPNYADSVKGR 9 EGVRYGDSWYDGDY 11 C#19 GDISSFNAMG 7FISSGGRSKYTDSVKGR 10 GSVVSYETGNYYEPSNY 12 C#20 GSISSFNGMG 8FISSGGRSKYTDSVKGR 10 GSVLSYVTGNYYEPSDY 13

TABLE 3 Association (k_(on)) and dissociation (k_(off)) rate constantsfor the interaction of α-cobratoxin and V_(H)Hs during surface plasmonresonance. Clone K_(off) (s⁻¹) K_(on) (M⁻¹s⁻¹) K_(D) (nM) #2 1.9 × 10⁻⁴5.2 × 10⁵ 0.4 #43 6.8 × 10⁻³ 2.9 × 10⁵ 24 #19 1.0 × 10⁻² 5.8 × 10⁵ 25#20 1.7 × 10⁻³ 1.8 × 10⁶ 1

TABLE 4 Protein constituents of Naja kaouthia venom. Protein Name(Accession #) MW Toxic Dose Synonyme Names (Da) LD₅₀ Function Referencesα-Cobratoxin 7820 0.1 mg/kg Inhibits neuromuscular Karlsson, 1973(P01391) i.v. transmission by binding to Long neurotoxin 1, Neurotoxin3, α- nicotinic acetylcholine receptors Cbtx at neuromuscular junctions.Cobrotoxin 9262 0.325 mg/kg  As above Meng et al., 2002 (P60771)(precursor) i.p. CBT, Short neurotoxin 1, NT1 Cobrotoxin II 6862 UnknownAs above Cheng et al., 2000 (P82849) CBT II, CBT2, Short neurotoxin 5Cobrotoxin-b 6944 400 mg/kg  As above Meng et al., 2002; (P59275) i.p.Cheng et al., 2000 CBT-b, Short neurotoxin 3, NT3 Cobrotoxin-c 6859  80mg/kg As above Meng et al., 2002; (P59276) i.p. Cheng et al., 2000CBT-c, NT2, Short neurotoxin 2 Short neurotoxin I 6983 Unkown As aboveChiou, S. H., et al., 1989 (P14613) Toxin C-6 Phospholipase A2 isozyme 116,271  10 mg/kg Catalyzes the hydrolysis of the Joubert and Taljaard,(P00596) i.v., mouse acyl group attached to the 2- 1980a; NnkPLA-I,CM-II position of 3-sn- Chuman et al., 2000 phosphoglyceridesPhospholipase A2 isozyme 2 16,016 4.4 mg/kg As above Joubert andTaljaard, (P00597) i.v., mouse 1980a; NnkPLA-II, CM-III Chuman et al.,2000 Cytotoxin 1 6701  1.3 mg/kg, Cytolytic activity Joubert andTaljaard, (P60305) i.v. 1980b; Cardiotoxin F-8, CTX1, CM-6 Fryklund andEaker, 1975; Ohkura et al, 1988 Cytotoxin 2 6745 1.2 mg/kg As aboveJoubert and Taljaard, (P01445) i.v. 1980b Cytotoxin CM-7A Cytotoxin 36708 1.2 mg/kg As above Joubert and Taljaard, (P01446) i.v. 1980b;Ohkura, et al., 1988 Cytotoxin CM-7, CX3, CT3 Cytotoxin IV 6739 1.48mg/kg  As above Ohkura et al., 1988; (P60303) i.p. Chiou et al., 1989Cytotoxin 5 6646 Unknown As above Ohkura et al., 1988 (P24779) CytotoxinII Cytotoxin like basic protein 6977 Unknown Low cytotoxic activityInoue et al, 1987 (P14541) Hemorrhagic 44493 Unknown Cleaves the vonWillebrand factor Ito et al., 2001 metalloproteinase- in humans, therebyinhibiting disintegrin kaouthiagin platelet aggregation during (P82942)hemorrhages Cobra venom factor 149,000 Unknown Compliment-activatingfactor of Eggertsen et al., 1981 (Q91132) venom Fritzinger et al., 1994;CVF. Complement C3 homolog Kock et al., 2004 Muscarinic toxin-like 7361Unknown Binds weakly to muscarinic Kukhtina et al., 2000 protein 1acetylcholine receptor (P82462) MTLP-1 Muscarinic toxin-like 7293Unknown As above Kukhtina et al., 2000 protein 2 (P82463) MTLP-2Muscarinic toxin-like 7615 Unknown As above Kukhtina et al., 2000protein 3 (P82464) MTLP-3 Weak toxin CM-9a 7438  82 mg/kg Binds weaklyto the nicotinic Joubert and Taljaard, (P25679) i.v. acetylcholinereceptor 1980c cited in the Universal Protein Resource website(UniProt). Weak tryptophan- 7613 Approximately 300 Binds weakly to thenicotinic Utkin et al., 2001 containing neurotoxin less potent than α-acetylcholine receptor (P82935) Cbtx and NT 2 WTX Cysteine-rich venom23621 Non-toxic Unknown Osipov et al., 2005 protein 23 (P84808) CRVP-23kCysteine-rich venom 24080 As above As above Osipov et al., 2005 protein24 (P84803) CRVP-24k Cysteine-rich venom 24093 As above As above Osipovet al., 2005 protein 25 (P84805) CRVP-25k

TABLE 5 Nucleotide and Amino Acid Sequences for Anti-α-cobratoxin V_(H)HC2, C19, C20 and C43. Nucleotide Sequences V_(H)H C2 (SEQ ID NO: 14)CAGGTAAAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCTGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTCTAGTATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAAGTGGTCGCAGTTATTACTAATGGTAATAGTCCAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTCGAGGGTGTTCGGTACGGTGATAGCTGGTACGATGGTGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA V_(H)H C19 (SEQ ID NO: 15)CAGGTAAAGCTGGAGGAGTCTGGGGGAGGTTTGGCGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTATAGGGTCTGGAGACATCTCCAGCTTCAATGCCATGGGCTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAATTGGTCGCATTTATTAGTAGCGGTGGTCGCTCAAAATATACAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGATCGACCTGAAACCTGAGGACACAGCCGTCTATTACTGTAATGCAGGTTCGGTGGTATCATACGAAACTGGTAATTACTACGAACCATCTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA V_(H)H C20 (SEQ ID NO: 16)CAGGTAAAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGGGTCTGGAAGCATCTCCAGCTTCAATGGCATGGGCTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAATTGGTCGCATTTATCAGTAGTGGTGGTCGCTCAAAATATACAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAGCACGGTGTATCTGCAAATGATCAACCTGAAACCTGAGGACACAGCCGTCTATTACTGTAATGTCGGTTCCGTGCTATCATACGTAACTGGTAATTACTACGAACCATCTGATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA V_(H)H C43 (SEQ ID NO: 17)CGGGTAAAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCTGTCTCTGGATCTACTTTCGATGATTATGCCATAGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAAGTGGTCGCAGTTATTACTAATGGTAATAGTCCAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTCGAGGGTGTTCGGTACGGTGATAGCTGGTACGATGGTGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA Predicted amino acid sequences V_(H)H C2(SEQ ID NO: 1)QVKLEESGGGLVLPGGSLRLSGAASGSISSIYAMGWYRQAPGKQREVVAVITNGNSPNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVEGVRYGDSWYDGDYWGQGTQVTVSS V_(H)H C19(SEQ ID NO: 2)QVKLEESGGGLAQAGGSLRLSCIGSGDISSFNAMGWYRQVPGKQRELVAFISSGGRSKYTDSVKGRFTISGDNAKNTVYLQMIDLKPEDTAVYYCNAGSVVSYETGNYYEPSNYWGQGTQVTVSS V_(H)H C20(SEQ ID NO: 3)QVKLEESGGGLVQPGGSLRLSCVGSGSISSFNIGMGWYRQVPGKQRELVAFISSGGRSKYTDSVKGRFTISGDNAKSTVYLQMINLKPEDTAVYYCNVGSVLSYVTGNYYEPSDYWGQGTQVTVSS V_(H)H C43(SEQ ID NO: 4)RVKLEESGGGLVQAGGSLRLSCAVSGSTFDDYAIGWYRQAPGKQREVVAVITNGNSPNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVEGVRYGDSWYDGDYWGQGTQVTVSS

TABLE 6 Identity and Similarity Among Amino Acid Sequences for V_(H)HC2, C19, C20 and C30. Identity similarity C2 C19 C20 C43 C2 100%/100%71% 73% 92% C19 76% 100%/100% 91% 68% C20 79% 95% 100%/100% 69% C43 94%75% 76% 100%/100% % identity is shown above the diagonal; % similarity,below.

TABLE 7 Consensus Sequence for V_(H)H C2, C19, C20 and C30. VHH_C2QVKLEESGGGLVLPGGSLRLSCAASGSISSIYAMGWYRQAPGKQREVVAVITNGNSPNYA 60 VHH_C43RVKLEESGGGLVQAGGSLRLSCAVSGSTFDDYAIGWYRQAPGKQREVVAVITNGNSPNYA 60 VHH_C19QVKLEESGGGLAQAGGSLRLSCIGSGDISSFNAMGWYRQVPGKQRELVAFISSGGRSKYT 60 VHH_C20QVKLEESGGGLVQPGGSLRLSCVGSGSISSFNGMGWYRQVPGKQRELVAFISSGGRSKYT 60CONSENSUS :**********. .********  **.  .  .:*****.******:**.*:.*. .:*:VHH_C2 DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVEGVR---YGDSWYDGDYWGQGTQ 117VHH_C43 DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVEGVR---YGDSWYDGDYWGQGTQ 117VHH_C19 DSVKGRFTISGDNAKNTVYLQMIDLKPEDTAVYYCNAGSVVSYETGNYYEPSNYWGQGTQ 120VHH_C20 DSVKGRFTISGDNAKSTVYLQMINLKPEDTAVYYCNVGSVLSYVTGNYYEPSDYWGQGTQ 120CONSENSUS ********** ****.****** .************. .*     *: :  .:*******VHH_C2 VTVSS 122 VHH_C43 VTVSS 122 VHH_C19 VTVSS 125 VHH_C20 VTVSS 125 C*****

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1. A method of obtaining a V_(H)H library comprising: (1) immunizing acamelid with whole venom or an extract thereof; (2) isolating nucleicacid sequences encoding the variable heavy fragment from the immunizedcamelid; and (3) transforming a suitable host with the nucleic acidsequences to prepare a recombinant V_(H)H library comprising V_(H)Hantibody fragments that can bind to one or more proteins in the venom.2. The method according to claim 1, wherein the camelid is a llama. 3.The method according to claim 1, wherein the venom is snake venom.
 4. Anisolated V_(H)H antibody fragment obtained from the library according toclaims
 1. 5. The isolated V_(H)H antibody fragment according to claim 4that can bind to one or more proteins in a venom.
 6. The isolated V_(H)Hantibody fragment according to claim 4, wherein the venom is snakevenom.
 7. The isolated V_(H)H antibody fragment according to claim 6,wherein the V_(H)H antibody fragment binds to α-cobratoxin.
 8. Anisolated heavy chain complementarity determining region (CDR) selectedfrom: an isolated heavy chain complementarity determining region 1(CDR1) comprising the amino acid sequence selected from SEQ ID NOS:5-8and 31 or a variant thereof; an isolated heavy chain complementaritydetermining region 2 (CDR2) comprising the amino acid sequence selectedfrom SEQ ID NO:9-10, 18, 30 and 33 or a variant thereof; and an isolatedheavy chain complementarity determining region 3 (CDR3) comprising theamino acid sequence selected from SEQ ID NOS:11-13, 29 and 32 or avariant thereof.
 9. An isolated V_(H)H antibody fragment comprising oneor more of the isolated CDR1 sequences and/or one or more of theisolated CDR2 sequences and/or one or more of the isolated CDR3sequences according to claim 8; or a variant thereof.
 10. An isolatedV_(H)H antibody fragment comprising the amino acid sequence of SEQ IDNO:1 or a variant thereof; the amino acid sequence of SEQ ID NO:2 or avariant thereof; the amino acid sequence of SEQ ID NO:3 or a variantthereof; or the amino acid sequence of SEQ ID NO:4 a variant thereof.11-13. (canceled)
 14. An isolated nucleic acid sequence encoding: theheavy chain complementarity determining region 1 (CDR1); the heavy chaincomplementarity determining region 2 (CDR2); and the heavy chaincomplementarity determining region 3 (CDR3); according to claim 8, or avariant thereof.
 15. An isolated nucleic acid sequence encoding theV_(H)H antibody fragments comprising the amino acid sequences accordingto claim 10 or a variant thereof.
 16. An isolated nucleic acid sequenceencoding the V_(H)H antibody fragment comprising the nucleic acidsequence of SEQ ID NO:14 or a variant thereof; the nucleic acid sequenceof SEQ ID NO:15 or a variant thereof; the nucleic acid sequence of SEQID NO:16 or a variant thereof; or the nucleic acid sequence of SEQ IDNO:17 or a variant thereof. 17-25. (canceled)
 26. A pharmaceuticalcomposition comprising an effective amount of the CDR according to claim8 and/or the V_(H)H antibody fragment according to claim 9 in admixturewith a suitable diluent or carrier.
 27. (canceled)
 28. A kit comprisingan effective amount of the V_(H)H antibody fragment according to claim 9together with ancillary agents and instructions for use thereof.
 29. Thekit according to claim 28, wherein the instructions for use thereofcomprise treating a subject exposed to a venom and/or treatingenvenomation in a subject and/or neutralizing a venom in a subject thathas been exposed to the venom.
 30. A method of treating a subjectexposed to a venom comprising administering an isolated V_(H)H antibodyfragment according to claim 10 to the subject in need thereof.
 31. Themethod of claim 30 wherein the venom is snake venom
 32. The methodaccording to claim 31 wherein the V_(H)H antibody fragment binds toα-cobratoxin.